The relationship between primary colorectal cancer histology and the histopathological growth patterns of corresponding liver metastases

Background The histopathological growth patterns (HGPs) are a prognostic and predictive biomarker in colorectal cancer liver metastasis (CRLM). This study evaluates the relationship between the HGP and primary colorectal cancer (CRC) histopathology. Methods A total of 183 treatment-naive patients with resected CRC and CRLM were included. Thirteen CRC histopathology markers were determined and compared between the desmoplastic and non-desmoplastic HGP; tumour sidedness, pT&pN stage, tumour grade, tumour deposits, perineural- (lympho-)vascular- and extramural venous invasion, peritumoural budding, stroma type, CRC growth pattern, Crohn’s-like lymphoid reaction, and tumour-infiltrating lymphocyte (TIL) density. Logistic regression analysis was performed using both CRC and CRLM characteristics. Results Unfavourable CRC histopathology was more frequent in non-desmoplastic CRLM for all markers evaluated, and significantly so for a lower TIL density, absent Crohn’s-like lymphoid reaction, and a “non-mature” stroma (all p < 0.03). The cumulative prevalence of unfavourable CRC histopathology was significantly higher in patients with non-desmoplastic compared to desmoplastic CRLM, with a median (IQR) of 4 (3–6) vs 2 (1–3.5) unfavourable characteristics observed, respectively (p < 0.001). Multivariable regression with 9 CRC histopathology markers and 2 CRLM characteristics achieved good discriminatory performance (AUC = 0.83). Conclusions The results of this study associates primary CRC histopathology with the HGP of corresponding liver metastases. Supplementary Information The online version contains supplementary material available at 10.1186/s12885-022-09994-3.


Tumour deposits (TD)
Definition: Tumour deposits are defined as discrete tumour nodules of any shape, contour or size that lack associated lymph node tissue, vascular structures or neural structures found within the lymph drainage area of the primary carcinoma. (1) Examination: AJCC 8 clarifies the interpretation of discrete tumour nodules found within the lymph drainage area of a primary colon or rectal carcinoma. Nodules containing no identifiable lymph node tissue or vascular/neural structures should be considered tumour deposits. The shape, contour, and size of the deposit are not considered in these designations. (1) The presence of tumour deposits is evaluated and the number of tumour deposits is counted.

Examination:
At least 3 blocks of tumour (optimally 5 or more blocks) should be submitted. A single H&E stained section from each block should be examined for venous or lymphatic vessel invasion. (3)

Definition:
Extramural vascular/venous invasion (EMVI) is defined as the presence of large vessel invasion beyond the muscularis propria. (5) Examination: Vessels with a clear endothelial lining and thin walled should be considered lymphatic (small), whereas large vessels (venous) includes all with a muscular wall. In suspicious cases, sections at multiple levels or sections with elastin staining have to be used to confirm venous invasion. Venous invasion beyond the muscularis propria is considered extramural vascular invasion. (5)

Photo:
A: EMVI in a large vein. B: Higher magnification of A, with highlight to vein wall (arrow). C: EMVI in a smaller vein. (5) Perineural invasion (PNI)

Definition:
Perineural invasion (PNI) is assessed positive when cancer cells are observed inside the nerve sheath, or when at least 33% of the nerve periphery is surrounded by cancer cells. (6) Examination: See definition above. When perineural invasion in found in any slide, it is considered positive.

Photo:
A: Tumor cells located inside the nerve sheath. B: Glandular element inside the nerve sheath. C and D: Nerves with at least 33% of the nerve periphery surrounded by cancer cells. (6) Peritumoural budding (PTB)

Definition:
Peritumoural budding (PTB) is defined as a single tumour cell or a cell cluster of up to 4 tumour cells at the tumour front. (7) Examination: See flowchart (figure 4) below. The prodecure is followed proposed by the ITBCC 2016. (7) Define the field (specimen) area for the 20x objective lens of the microscope based on the eyepiece field number (FN) diameter. Select the H&E slide with greatest degree of budding at the invasive front. Scan 10 individual fields at medium power (10x objective) to identify the 'hotspot' at the invasive front. Count tumour buds in the selected 'hotspot' (20x objective). Divide the bud count by the normalization factor to determine the tumour bud count per 0,785 mm 2 .

Normalization factor depending on eyepiece field number (FN) diameter:
Objective Expanding growth pattern (EGP)

Definition:
Colorectal carcinomas are defined as 'expanding' when the invasive margin is pushing or reasonably well circumscribed. (8)

Examination:
The invasive front of the primary cancer is classified as 'expanding growth pattern' (EGP) based on the predominant morphology, as defined by Jass et al, where the 'expanding' type had been described as the pushing growth type of adenocarcinoma. (8) The amount of EGP present at the invasive front is noted in percentage of the total invasive front.

Photo:
Colorectal carcinoma with an expanding growth pattern where the tumour pushes the surrounding stroma with a sharp dividing interface. (9) Infiltrating growth pattern (IGP)

Definition:
Colorectal carcinomas are defined as 'infiltrating' when the tumour invaded in a diffuse manner with widespread penetration of normal tissues.

Examination:
The invasive front of the primary cancers were classified as infiltrating growth pattern (IGP) based on the predominant morphology, as defined by Jass et al, where the infiltrating type had been described as the wide spread streaming form of adenocarcinoma. (8) The amount of IGP present at the invasive front is noted in percentage of the total invasive front.

Photo:
Colorectal carcinoma with an infiltrating growth pattern where tumour glands penetrate the surrounding stroma with an irregular interface. (9) Stroma type

See flowchart below for interpretation.
Examples of the different types of stroma identified at the extramural front. (10) Immature stroma type (type A)

Definition:
Tumours with a myxoid stroma are defined as ' immature'. (11) Examination: The type of stroma is classified based on the predominant morphology of the stroma, where 'immature' (myxoid) stroma is defined as stroma with an amorphous mucinous substance, typically composed of a slightly basophilic or amphophilic, vacuolated extracellular material among the collagen fibers present in at least one field as viewed through a 40x objective lens. (11)

Definition:
Tumours without a myxoid stroma, but with a keloid-like collagen in the fibrotic stroma was defined as 'intermediate'. (11) Examination: The type of stroma is classified based on the predominant morphology of the stroma, where 'intermediate' (keloid-like collagen) stroma is defined as hyalinized thick bundles of hypocellular collagen with bright eosinophilic hyalinization. (11)

Definition:
Tumours that were only composed of fine mature collagen fibers stratified into multiple layers in all reactive fibrous zones were defined as 'mature'. (11) Examination: The type of stroma is classified based on the predominant morphology of the stroma, where 'mature' stroma is defined as fine mature collagen fibers stratified into multiple layers in all reactive fibrous zones. (11) Photo: Mature stroma type, characterized by only fine mature collagen fibres stratified into multiple layers. This stroma type does not contain myxoid stroma or keloid-like collagen. (10) Crohn's-like lymphoid reaction (CLR)

Definition:
Crohn's-Like Lymphoid Reaction (CLR) is defined as the presence of peritumoural lymphoid aggregates (LAs). (12) For evaluation we use the presence of the LAs and determine the number of LAs.

Examination:
We used the evaluation method of Ueno et al (12). Nodular lymphoid aggregates lining the tumour periphery are evaluated with regard to the CLR status (Photo 1). The following are not evaluated as LAs associated with CLR: 1. LAs from mucosa-associated lymphoid tissue (i.e. located within the mucosal layer or just below the muscularis mucosae). 2. LAs judged to be part of small lymph nodes rather than LAs associated with CLR on the basis of the existence of circumferential connective tissue around LAs. 3. Non-nodular LAs, including irregularly shaped as well as long and narrow ones. Small LAs are regarded as those in which the number of lymphocytes appeared to increase in comparison to those in the background but which had no definite 'aggregation', so a cut-off of 300 micro meter or larger in size is used. The largest LA in each patient is identified and its maximum diameter is determined with a calibrated ocular reticule. The outline of LAs for measurement is determined under low-power magnification.

Definition:
Tumour infiltrating lymphocytes (TIL) are determined as a percentage of mononuclear inflammatory cells over total intratumoural stromal area and counted in 5 HPF (total magnification, ×200-400) in the invasive front or areas surrounding the deposits, except for tumour areas with crush artifacts, necrosis or regressive hyalinization (14).

Examination: See flowchart (figure 3) below.
The tumour area at low magnification is selected and the percentage of the area that is filled with mononuclear cells in the stromal area around the tumour border is assessed at high magnification (×200). Mononuclear cells are defined, including the lymphocytes in the stromal area, as TILs. Granulocytes and other polymorphonuclear leukocytes are excluded (14). The recommendation of the International TILs Working Group for evaluating the density is used (15).

Photos:
Examples of the evaluation of tumour infiltrating lymphocytes (TIL). The mean percentage of the area occupied by TILs in the intratumoural stroma is reported in each section. (14) Standardized approach for TILs evaluation in breast cancer. (15) Different percentages of TILs in breast cancer (15).